Volumetric characterizations of protein denaturation and ligand binding

grantor: University of TorontoWe have used model proteins to study the role of water in two important protein recognition processes. In the first project, we studied the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentration. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein assumes its unfolded conformation with disrupted secondary and tertiary structures. Increasing urea concentration to 1.5 M induces further unfolding. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, [Delta][nu], accompanying the acid-induced denaturation of SNase increases. At all urea concentrations, the partial specific adiabatic compressibility, 'k 'os, of the protein decreases upon its unfolding. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80% of that expected for the fully extended conformation. In the second project, we characterized the binding of ribonuclease A (RNase A) to cytidine 2'-monophosphate (2'-CMP) and cytidine 3'-monophosphate (3'-CMP) at different temperatures. Our results suggest that RNase A undergoes some structural alteration upon binding to either ligands. The volume and compressibility results suggest that 210 ± 40 water molecules are released to the bulk state upon the binding of 2'-CMP or 3 '-CMP to RNase. The binding of these ligands leads to a ~15% decrease in the configurational entropy of the protein, consistent with a decrease in its conformational dynamics upon ligand binding. Thus, RNase A becomes "dehydrated", "smaller", "tighter", and "less dynamic" upon binding either ligand.M.Sc